Recombinant β-glycosidases from hyperthermophilic Sulfolobus solfataricus (SsβGly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70°C or greater. Compared with SsβGly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80°C and pH 5.5 being 22 days. The stability of CelB but not that of SsβGly is decreased 4-fold in the presence of 200 mM lactose at 80°C. CelB displays a broader pH/activity profile than SsβGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95°C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki ≈ 0.3 M), and at 80°C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsβGly, respectively. SsβGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl β-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsβGly, compared with CelB. Overall, CelB and SsβGly share their catalytic properties with much less thermostable β-glycosidases and thus seem very suitable for lactose hydrolysis at ≥70°C. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 322–332, 1999.